LIPC) is a glycoprotein in the triacylglycerol lipase family and mainly synthesized in and secreted from the liver. Previous studies demonstrated that hepatic lipase is crucial for reverse cholesterol transport and modulating metabolism and the plasma levels of several lipoproteins. This study was conducted to investigate the suppression effect of high-density lipoprotein cholesterol (HDL-C) levels in a genome-wide association study and explore the possible mechanisms linking triglyceride (TG) to LIPC variants and HDL-C. Genome-wide association data for TG and HDL-C were available for 4657 Taiwan-biobank participants. The prevalence of haplotypes in the LIPC promoter region and their effects were calculated. The cloned constructs of the haplotypes were expressed transiently in HepG2 cells and evaluated in a luciferase reporter assay. Genome-wide association analysis revealed that HDL-C was significantly associated with variations in LIPC after adjusting for TG. Three haplotypes  in LIPC were identified. H2: CTA was significantly associated with HDL-C levels and H1: TCG suppressed HDL-C levels when a third factor, TG, was included in mediation analysis. The luciferase reporter assay further showed that the H2: CTA haplotype significantly inhibited luciferase activity compared with the H1: TCG haplotype. In conclusion, we identified a suppressive role for TG in the genome-wide association between LIPC and HDL-C. A functional haplotype of hepatic lipase may reduce HDL-C levels and is suppressed by TG.Hepatic lipase (encoded by  LIPC), a glycoprotein in the triacylglycerol lipase family, which participates in the hydrolysis of triglycerides (TGs) and phospholipids, is mainly synthesized and secreted from the liver [Hepatic lipase  and lipid levels were mainly significant with HDL-C [LIPC promoter SNP C-514 T/rs1800588 with TG and HDL-C levels were inconsistent [Previous GWAS in various ethnic populations showed that the associations between th HDL-C ,12,13. Hnsistent . Numerounsistent ,17,18. SMediation analysis is a third variable effect model for investigating causal processes from the effect of the independent variable to the dependent variable . This meLIPC) variants and HDL-C levels [Our previous candidate gene studies involving mediation analysis in a general Taiwanese population provided the first evidence of the suppression effect ,25. ReceC levels , identifLIPC as well as estimate their effect on lipid-proteins, along with HDL-C and TG levels. We also estimated the functional activity of LIPC haplotypes to provide further evidence of the presence of genetic factors responsible for the cardiovascular risk associated with lipid profile in Taiwanese populations.The primary objective of this study was to validate the suppressive effect of mediation analysis in GWAS and identify haplotype blocks in The study cohort for the GWAS consisted of participants from the Taiwan Biobank (TWB), which gathered information from recruitment centers across Taiwan between 2008 and 2015 . A totalTM-TWB genome-wide array including 642832 SNPs, with the help of the National Center of Genome Medicine of the Academia Sinica. All samples enrolled in the present analysis had a call rate of ≥97%. QC for SNPs was established as follows: SNP call rate <3%, minor allele frequency <0.05 and violation of Hardy–Weinberg equilibrium  were excluded from subsequent analyses. After QC, 4000 participants and 614,821 SNPs were evaluated in the GWAS analysis.The Taiwan Biobank study group designed the TWB genotype array for a high-throughput Affymetrix Axiom genotyping platform. SNPs on the Axiom Genome-Wide CHB 1 Array plate with 5% minor allele frequencies in a set of 4675 samples from Taiwanese population, previously genotyped at the National Center of Genome Medicine of the Academia Sinica, Taipei, Taiwan, were selected for the new TWB array. The genomic DNA was genotyped using the AxiomFor TWB participants, DNA was isolated from blood samples using a QIAamp DNA blood kit  following the manufacturer’s instructions. SNP genotyping was performed using custom TWB chips and run on the Axiom Genome-Wide Array Plate System .2).The following clinical phenotypes were analyzed: body height, body weight, body mass index (BMI), waist circumference, waist-to-hip ratio and systolic and diastolic blood pressure. Biochemistry data enrolled for analysis included fasting plasma glucose, as well as lipid profiles including total cholesterol, HDL-C, low-density lipoprotein cholesterol (LDL-C) and TG levels. BMI was calculated as body weight (kg)/height  to phase the genotypes and to evaluate the haplotypes of each individual for rs1077834, rs1800588 and rs2070895. The frequency of each haplotype was calculated and only those with frequencies greater than 1% in the cohort were further analyzed. The haplotype association for serum HDL-C levels was tested with linear regression models under the assumption of an additive genetic model and corrected for the effects of age, sex, BMI and smoking, with or without TG.LIPC haplotype and HDL-C, a mediation model was assumed , were subsequently extracted by XhoI and HindIII digestion and gel-purification. The inserts were further subcloned into the pGL4.10 (Luc 2) vector , upstream of the firefly luciferase reporter gene. The correctness and direction of the inserts in relation to the luciferase gene were evaluated by sequencing.A genomic DNA fragment of the on study . The seq2 at 37 °C.HepG2 cells were cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal calf serum  in 5% COLIPC 5′-promoter constructs (500 ng) were co-transfected with 50 ng pRL-TK (Promega), which encoded Renilla luciferase into HepG2 cells using X-tremeGENE HP DNA Transfection Reagent . Luminescence was quantified by using a dual-luciferase reporter assay system (Promega) according to the manufacturer’s protocol after 24 h transfection. All experiments were performed in triplicate and data for firefly luciferase activity were normalized to that of the Renilla luciferase for each sample.Cells were plated in a 6-well plate and grown to 80–90% confluence. The t-test or analysis of variance. A chi-square test or chi-square test for the trend was used to examine differences in the distribution of categorical data. TG and HDL-C levels were logarithmically transformed before analysis to adhere to a normality assumption. A generalized linear model was used to analyze TG and HDL-C levels in relation to the predictors of the investigated genotypes and confounders. The genetic effect was considered as additive and adjusted for age, sex, BMI and smoking status. Genome-wide scans were calculated using PLINK. P values below the threshold of p = 5 × 10−8 were considered significant. All calculations were performed using SPSS version 22 . P values <0.05 using a two-sided test were considered significant. Missing data were managed using listwise deletion. The analysis of deviation from the Hardy Weinberg equilibrium, estimation of linkage disequilibrium between polymorphisms, association between haplotypes and lipid-protein levels were performed using the Golden Helix SVS Win32 7.3.1 software .The clinical characteristics of continuous variables are expressed as the means ± standard deviations, which were tested using a two-sample p < 0.001). In addition, men showed significantly higher BMI (p < 0.001), waist circumference (p < 0.001), waist/hip ratio (p < 0.001) and circulating levels of LDL-C (p = 0.006) and TG (p < 0.001) than women. In contrast, circulating HDL-C (p < 0.001) and total cholesterol levels (p = 0.015) were lower in men than in women.Demographic data and lipid profiles of the participants, grouped by sex, are summarized in CETP (p = 10−8–10−27) (LIPC (LIPC (p = 1.41 × 10−15) , indicating that the association between LIPC variants and HDL-C is strongly suppressed by TG. Linkage disequilibrium analysis showed that rs261334 was in strong linkage disequilibrium (r2 = 0.96) with the promoter SNP at rs1800588, which is known to function in regulating LIPC. Our candidate gene study also showed that rs1800588 is strongly associated with TG and HDL-C levels. Therefore, rs1800588 was used to replace rs261334 in subsequent analysis.After performing QC following the standard procedure, the results showed that several variants in a region of chromosome 16 had a highly significant association with HDL-C levels. These significant variants were upstream and in the 5′-untranslated region of 8–10−27) A. After 7) (LIPC B. The mo× 10−15) C. The mo5) . Three major haplotypes were observed, accounting for 99.9% of all inferred haplotypes. After adjusting for age, sex, BMI and smoking, haplotype H2: CTA was found to be associated with significantly increased HDL-C levels (p = 0.0295). When further adjusted for TG, haplotypes H1: TCG and H2: CTA were found to be associated with significantly decreased (p = 0.0003) and increased (p = 0.0009) HDL-C levels, respectively. This indicates that the association of LIPC haplotypes with HDL-C was completely or partially mediated by TG  (criterion 1), which in turn presented significant negative effects on HDL-C (p = 9.17 × 10−48) (criterion 2). The total effect of H1: TCG on HDL-C was −0.021 (p = 0.05) (criterion 3). The results of the Sobel test showed a z value of 2.38 and p value of 0.023 (criterion 4). Furthermore, the direct effect (γ’) of H1: TCG on HDL-C (-0.033) was larger than the total effect (αβ+γ’) (0.021) and had the reverse sign with the mediation effect (αβ) (0.012), demonstrating a suppression effect in this model.Four criteria were evaluated to establish mediation and suppression effects . After ap = 1.8 × 10−5, p = 0.0003 and p = 5.0 × 10−5, respectively). In addition, haplotype H2: CTA showed lower luciferase activity than the major haplotype H1: TCG (p = 0.006) and rare haplotype H3: CCA (p = 0.005), respectively , luciferase expression was significantly greater following transfection with haplotypes H3: CCA, H2: CTA and H1: TCG  gene, was found to be strongly associated with HDL-C. This result is consistent with those of previous studies. CETP is an important protein that modulates HDL metabolism and genetic variants in CETP have been shown to be associated with HDL-C levels [This study investigated the suppressive role of TG in a GWAS between C levels . The proC levels . CETP isC levels ,35.CETP and HDL-C, the effect of LIPC promoter variants on HDL-C is very variable [LIPC polymorphisms and HDL-C [LIPC were significantly associated with HDL-C only after adjusting for TG. LIPC codes for the synthesis of HL, which participates in HDL-mediated reverse cholesterol transport, carrying the TGs and cholesterols from the circulating blood to the liver and is involved in the hydrolysis of TGs [LIPC and HDL-C.Unlike the consistent results indicating an association between variable ,37,38,39s of TGs . Thus, TLIPC, we identified three haplotypes in the section of the proximal LIPC promoter that included three common polymorphisms . Haplotype analysis demonstrated that the common haplotype H1: TCG was significantly associated with TG and mediated the association with HDL-C. However, the minor haplotype H2: CTA lost significant association with TG, though possessing increased significant association with HDL-C. Further, the rare haplotype H3: CCA subsequently recovered the significant association with TG but lost the significant association with HDL-C, irrespective of further adjustments for TG. These findings are comparable to those of previous studies, which reported that the -514T and -250A variants (SNP2 and SNP3) in H2: CTA are associated with elevated HDL-C and TG [LIPC H2: CTA is the major factor determining plasma HDL-C levels. The effect of the SNP2 C allele may only become apparent in combination with the SNP1 T/C and SNP3 G/A in H1: TCG and H3: CCA.To investigate the causality of SNPs in the promoter region of C and TG ,40,41. TLIPC and HDL-C, we performed mediation analysis to validate the suppression effect. To the best of our knowledge, this is the first evidence of a suppression effect in large-scale GWAS. According to our data, we try to explain the suppression effect more detail by using a pattern with signs and directions  between the TG level (upper red arrow) and HDL-C level (right red arrow), which may eventually produce the opposite effect  to partially reverse the HDL-C level and therefore masks the significance (red square) between LIPC haplotype H1: TCG and HDL-C into non-significance (blue square).To explore the possible mechanism of the effect of TG on the association between rections . As carrLIPC promoter [LIPC and decrease promoter activity [The luciferase reporter assay further indicated that the haplotypes were functional and the functional activity of the common haplotype H1: TCG was reduced by the minor haplotype H2: CTA involving the HDL-C increasing alleles -514T and -250A. Several regulatory elements have been reported in the proximal region (from −1600 to +129) of the promoter ,43,44. Apromoter ,46. The LIPC variants and HDL-C. This suppression effect is due to the multiple functions of HL and CETP in lipid metabolism. HL mainly participates in the hydrolysis of TG from intermediate-density lipoproteins and not from very low-density lipoprotein particles [LIPC regulation, it remains very important to establish an animal model to confirm this suppressive effect in vivo.The suppression effect is seldom applied in genetic associations. In the present study, we replicated the suppression effect in a second independent Taiwanese population. By combining the results of haplotype analysis and functional activity, we hypothesized that TG levels mediate the suppression of the association between articles ,48 and rarticles . The TG trans- or cis-regulation in the distal region should be considered.One limitation of our study is that we did not measure HL activity. We predicted that the haplotypes affect the activity of HL and thereby influence plasma HDL-C. Another limitation is that several studies have demonstrated the absence of mutations in the coding sequence of HL that would account for variance in HL activity ,49,50. HLIPC and HDL-C. We further found that the LIPC haplotype is functional. Therefore, the reduced HL activity observed in carriers of H2: CTA may occur because of decreased transcription. This molecular mechanism of gene regulation in the lipid profile should be further analyzed.In conclusion, we provide the first evidence of the suppressive role of TG in the genome-wide association between


This study aimed to investigate the anticancer properties of some potential probiotics Bifidobacterium. breve, 2. a single strain of Lactobacillus. reuteri, 3. a cocktail of 5 strains of Lactobacilli (LC), 4. a cocktail of 5 strains of Bifidobacteria (BC), 5. a cocktail of 10 strains from Lactobacillus and Bifidobacterium (L+B). Apoptosis rate, EGFR, HER-2 and PTGS-2 (COX-2 protein) expression levels were assessed as metrics of evaluating anticancer properties. Effect of BC, as the most effective group in vitro, was further assessed in mice models.Anticancer effects of potential probiotic groups were investigated following of in LS174T cancer cells compared to IEC-18 normal cells. 1. a single strain of EGFR by 4.4 folds, HER-2 by 6.7 folds, and PTGS-2 by 20 folds among the LS174T cells. In all these cases, BC did not interfere significantly with the expression of the genes in IEC-18 cells. This cocktail has caused only 1.1 folds decrease, 1.8 folds increase and 1.7 folds decrease in EGFR, HER-2 and PTGS-2 expression, respectively. Western blot analysis confirmed these results in the protein level. BC significantly ameliorated the disease activity index, restored colon length, inhibited the increase in incidence and progress of tumors to higher stages and grades.BC induced ~21% and only ~3% apoptosis among LS174T and IEC-18 cells respectively. BC decreased the expression of EGFR, HER-2 and PTGS-2 (COX-2), while having significant anti-CRC effects on CRC mice models. In general, this potential probiotic could be considered as a suitable nutritional supplement to treat and prevent CRC.BC was the most efficient treatment in this study. It had considerable “protective” anti-cancer properties and concomitantly down regulated  Lactobacillus and Bifidobacterium spp. Evidence from many studies suggest a preventive role for LAB probiotics in the onset of CRC both in vitro and in vivo [EGFR and HER-2 genes and proteins deregulate this pathway, leading to increased cell proliferation, prolonged survival, anti-apoptosis, and metastasis [PTGS-2 gene), the inducible form of an enzyme responsible for converting arachidonic acids into prostaglandins (PGEs) [Colorectal cancer (CRC) is the third most common type of cancer, being surpassed by only lung and breast cancers, and the second cause of cancer-related deaths worldwide . There a in vivo –8. Some  in vivo , 8, 9. O in vivo . Brieflytastasis –13. Henctastasis , 13. In s (PGEs) , 15. PGEs (PGEs) , 16, 17.Lactobacillus and Bifidobacterium spp. in vitro and in vivo.Several studies suggest the concurrent increase in the expression of COX-2 and EGFR , COX-2 aBifidobacterium. breve, 2. a single strain of Lactobacillus. reuteri, 3. a cocktail of 5 strains of Lactobacilli (LC), 4. a cocktail of 5 strains of Bifidobacteria (BC), 5. a cocktail of 10 strains from both Lactobacillus and Bifidobacterium (L+B).The anti-cancer effects of the 5 following treatment groups were investigated: 1. a single strain of Lactobacillus strains were from L. plantarum, L. rhamnosus, L. brevis, and L. reuteri species and the five Bifidobacterium strains were from B. bifidum, B. breve, B. longum species . The Bifidobacterium strains were isolated in our previous study from healthy mother's milk and their healthy infants’ stool [Lactobacillus strains, also isolated from our previous study [The five s’ stool  and the us study  were obtus study –23. All Although MRS is the medium which contains animal origin substances, this medium was only used for experimental analysis in this study and should be replaced by human compatible medium for production.2 at 37°C in high glucose DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin .The reason for choosing LS174T as the colon cancer cell line was based on the previous studies reporting simultaneously high expression levels of EGFR, HER2 and COX-2 in this cell line [The anticancer properties of the bacterial groups were assessed on 2 cell lines including the human colon adenocarcinoma cell line LS174T and the rat normal non-transformed intestinal cell line IEC-18  , a cocktail of 5 strains of Lactobacilli (LC), a cocktail of 5 strains of Bifidobacteria (BC), and a cocktail of the strains from both Lactobacillus and Bifidobacterium genera (L+B) were prepared. To make the bacterial cocktails equal amounts of each dilution were mixed into one tube. Cells (~3×105/ well) were seeded in DMEM/ 5% FBS inside 6-well culture plates and, after 72- hours plating , the desired concentration of the bacteria (based on the results of the MTT assay) were treated onto cells and incubated for 120 hours at 37°C in 5% CO2.All the bacterial strains were cultured in MRS broth  under anaerobic condition at 37°C for 16 hours. The strains were then centrifuged at 8000 × g for 5 minutes and the pellets were diluted in high glucose DMEM  containing 10% FBS . The bacterial concentrations  were established by measuring the OD5/ well) were treated with the desired concentration of the drugs (based on the results of the MTT assay) and incubated for 120 hours in the same conditions as for the bacterial treatment.The anti-EGFR and HER-2 monoclonal antibodies cetuximab  and trastuzumab  were used as controls in this study. These drugs were diluted in sterile distilled water to prepare the concentrations of 10, 20 and 30 μg/ml for each drug. Cells (~3×103 /well) were seeded in DMEM/ 5% FBS inside 96-well microplates and, after 24 hours plating, treated with different concentrations of bacteria  and drugs . Untreated cells were used as controls. The cells were incubated for 24 hours at 37°C in 5% CO2. The cell viability was then checked by reading the absorbance at 570 nm using ELISA microplate reader . The analysis for each group was repeated in triplicate. The following formula was used to calculate the percentage of proliferating cells inside each well:To find the best treatment dose, the anti-proliferative effects of the bacterial and drug treatments on LS174T and IEC-18 cells were evaluated by the MTT assay using the commercially available MTT kit , according to the manufacturer’s instructions. Both LS174T and IEC-18 cells (~5 × 105/ well) were seeded in DMEM/ 5% FBS inside 6-well culture plates and, after 72 hours incubation, were treated by the desired concentrations of the bacteria and drugs (based on the results of the MTT assay) and incubated for 120 hours at 37°C in 5% CO2. The cells were then harvested and washed twice with PBS, and following centrifugation, the cell pellets were re-suspended in Annexin binding buffer according to the manufacturer’s protocol. Cells were labeled by FITC-conjugated Annexin V and PI and analyzed on a flow cytometer  for the detection of Annexin V and PI positive subpopulations. The experiments were performed in duplicates and the data were analyzed using the Flow Jo software. Untreated cells were used as negative controls and cetuximab and trastuzumab were used as positive controls.Apoptosis, as a metric of evaluating anticancer properties of the bacterial groups, was determined by the Annexin V (FITC-conjugated)-Propidium iodide (PI) flow cytometric method using a commercially available kit , according to the manufacturer’s instructions. LS174T and IEC-18 cells , as well as untreated cells (as negative controls) were extracted using a total RNA extraction kit , according to the manufacturer’s instructions. The quality and quantity of the RNAs were assessed by measuring the absorbance at 230, 260 and 280 nm in a Thermo Scientific™ NanoDrop 2000 and 2000c UV ⁄ Visible Spectrophotometer. RNA integrity was determined by running the samples on a 2% agarose gel. cDNA templates were synthesized from 400 ng RNA using the PrimeScript cDNA synthesis kit , according to the manufacturer’s protocol. The quantification of the genes EGFR, HER-2 and PTGS-2 of the bacterial and drug- treated cells (relative to the untreated cells), were determined using SYBR Premix Ex Taq  and the primers retrieved from the online Primer-Bank website http://pga.mgh.harvard.edu/primerbank  housekeeping gene.The expression levels of imerbank . All ass7 cells treated with bacterial groups, drug groups (as positive controls), as well as untreated cells (as controls) using a total protein extraction kit , according to the manufacturer’s instructions. The Bradford assay was used to determine the total protein concentration, with the bovine serum albumin (BSA)  used as the standard protein. The sample concentrations were equalized and diluted in Tris-Glycine-SDS sample buffer containing 2-mercaptoethanol and methylene blue  and 20 μl was loaded in a 10% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE). Lysates were resolved by electrophoresis and transferred onto PVDF membranes . The membranes were blocked for 1 hour with 5% skimmed milk in PBS/0.1% Tween-20. Membrane were immunoblotted overnight at 4°C with anti-EGFR, anti-HER-2, anti-COX-2 rabbit , as well as anti-rabbit monoclonal β-Actin antibody (cat no: 4970), all diluted 1:1000 in 2.5% skimmed milk in PBS/0.1% Tween-20. After washing the membrane with PBS/ 0.1% Tween-20, the membrane was incubated for 1.5 hours with horseradish peroxidase-conjugated anti-rabbit IgG , diluted 1:1000 as other antibodies. After washing, enhanced chemiluminescence  was used for detection. Each experiment was repeated in triplicate. The protein bands were quantified by the ImageJ software.The expression levels of the onco-marker proteins EGFR, HER-2 and COX-2 were investigated using Western blot. Total protein was isolated from ~10in vitro experiment (BC) were further investigated in vivo. Female BALB/c mice (6–8 weeks old), obtained from Pasteur Institute of Iran, were kept in polycarbonate cages (5 mice per cage) under controlled environmental conditions at 22°C and 50% humidity in a 12 hours light/dark cycle. Enough food and water were made available to the animals. Mice were categorized into three groups: 1) PBS-gavaged negative control group, 2) Azoxymethane (AOM)/DSS + PBS positive control group, 3) AOM/DSS + BC in PBS test group. Five mice per group were used.The anticancer properties of the most effective bacterial treatment in 11 colony-forming units) BC in PBS. The orogastric gavage of PBS or BC was started a week before tumor induction by AOM/DSS and continued daily until sacrifice. To induce colon cancer, the second and third groups were intraperitoneally injected with a single dose of AOM (10 mg/kg) (Sigma-Aldrich) injection, as the carcinogenetic agent. After one week, water containing 2% DSS  was given to the second and third groups for 5 days , followed by 2 weeks of recovery, replacing the DSS water with regular water. The mice received another two cycles of water containing 2% DSS [A total of 0.2 mL PBS was administered daily to the first two groups, whilst the last group was orally administered with 0.2 mL  which was in accordance with the ethical standards of the Helsinki Declaration in 1975 and its later amendments.Sixty four days after AOM injection, all mice were sacrificed by rapid cervical dislocation, excised and opened longitudinally to remove their colons. Colons were cleaned with PBS and measured for length and number of tumors from the start of ceccum to the anus. Three sections (~1 cm) of rectum samples were recovered from each mouse, fixed in 10% formalin embedded in paraffin, flash-frozen in liquid nitrogen and stored at −80°C until analyzed for histology. The colon sectionswere sectioned at 3 μm by a microtome for hematoxylin-and-eosin (H&E) staining. The slides were analyzed at a magnification of ×100. Histological score was determined using a BX43 Olympus microscope in a blind manner and possible histological variations between the three groups were compared. Because this CRC model was associated with chronic intestinal inflammation, the level of DSS-causing inflammation was also assessed and compared between the three groups of mice.Data were expressed as mean ± SD. For comparison between two groups Student’s t-test and Fisher exact test and for comparison between multiple groups one-way ANOVA were used in Graphpad prism Version 8. A P-value ≤ 0.05 was considered as statistically significant.The MTT assay was used as an anti-proliferative assay to find the best treatment dose for the bacterial and drug groups for treating the cell lines. Untreated cells were used as controls. According to the results of the MTT assay , performApoptosis rate was assessed by flow cytometry as a metric of evaluating anticancer properties of the bacterial groups in this study. Untreated cells were used as negative controls and cetuximab and trastuzumab were used as positive controls.The flow cytometry results, performed in duplicates, showed that among the bacterial treatments, BC (20.5% primary apoptosis) had the highest effect on apoptosis induction after 120h incubation on LS174T cells (P = 0.03) .B. breve, L+B and L. reuteri induced 18.52%, 17.12%, 16.62%, and 11.12% primary apoptosis on LS174T cells after 120h, respectively  managed to have the least interruption on IEC-18 cells (P = 0.05).B. breve, L. reuteri and LC respectively induced 8.75%, 8.45%, 4.61% and 4.31% higher apoptosis rates among IEC-18 cells, compared to untreated control cells.L+B, Cetuximab and trastuzumab respectively induced 5.85% and 8.45% apoptosis among IEC-18 cells.EGFR, HER-2 and PTGS-2 genes, as onco-markers in colorectal cancer. In all assessments, untreated cells were used as negative controls and cetuximab and trastuzumab were used as positive controls. All the experiments were carried out in triplicates.Real time PCR was used to assess the expression levels of EGFR gene among LS174T cells during 120h incubation. However, the results were only significant in case of BC , as was also the case for cetuximab and trastuzumab .The results presented in EGFR down regulation .There was no significant difference between the effectiveness of the other 4 bacterial treatments in EGFR expression among the IEC-18 normal cells , whilst 2 groups increased its expression . These changes were only significant in case of LC , as was also the case for cetuximab and trastuzumab .Most bacterial groups decreased the HER-2 was much more pronounced among LS174T cells, compared to the EGFR gene. The reduction in the expression of HER-2 was significant for all the bacterial groups (P = 0.0001), similar to the drug groups (P = 0.001).Comparing Figs HER-2 by 6.7 folds but there was no significant difference between the effectiveness of all the other 4 bacterial groups in HER-2 down regulation .BC down regulated HER-2 by 9.1 folds (P = 0.05) and 6.7 folds (P = 0.09).Cetuximab and trastuzumab respectively down regulated HER-2 expression among the IEC-18 normal cells. This increase was only significant in case of LC , as was also the case for cetuximab and trastuzumab .All the bacterial groups increased PTGS-2 among LS174T is more considerably influenced by the bacterial groups, compared to the EGFR and, to a less extent, to the HER-2 genes. A very significant reduction is observed in the expression of PTGS-2 compared to the control, for all the bacterial groups (P<0.0001), similar to the cetuximab and trastuzumab .PTGS-2 expression) had the highest influence on down regulating PTGS-2, among the bacterial groups.BC . B. breve, although decreased PTGS-2 expression (by 1.3 folds), its result was not significant.All the bacterial groups, except PTGS-2 expression by 4.4 folds.Cetuximab and trastuzimab both decreased Western blot analysis was performed to evaluate the expression levels of the proteins EGFR, HER-2 and COX-2. Untreated cells were used as negative controls and cetuximab and trastuzumab were used as positive controls. All the experiments were performed in triplicates. Western blotting  showed tBC had no significant changes in the expression of these proteins among IEC-18 cells and the effect of LC among IEC-18 cells was only significant in case of COX-2 levels.Both drugs significantly changed the expression of EGFR, HER-2 and COX-2 among IEC-18 cells (P = 0.001).in vitro, and therefore its anticancer properties were further investigated in mice models. As shown in BC was the most effective treatment Tumors presented in BC-treated mice were also significantly smaller than those observed in untreated CRC mice (P = 0.001). BC-treated mice had significantly higher colon length and lower tumors incidence , compared to AOM/DSS CRC mice  (P = 0.04) . HistoloLactobacillus spp., from normal fecal samples, and five Bifidobacterium spp., from mother’s milk and infant stool, had different degrees of anti-proliferative, anti-pathogenic, antibacterial and antimicrobial activities [Probiotics have recently been suggested as interesting cancer preventive/ treating bacteria which exert their anti-cancer properties through different proposed mechanisms. Some limited studies have suggested interference of certain probiotics with the activation of EGFR family and COX-2 expression, the over expression of which are involved in many cancer types , 27–29. tivities , 23.In this study, BC (20.5% primary apoptosis) had the highest effect on inducing apoptosis after 120h incubation on LS174T cells among the bacterial treatments. Besides, BC managed to have the least interruption on IEC-18 cells (~3% apoptosis rate). All these results prove the very effective apoptotic feature of BC compared to other bacterial treatments, with a “protective” property despite a high ability of apoptosis induction in cancer cells.B. breve were the next functional candidates among the potential probiotics tested, which had reasonable apoptotic effects on LS174T cells whilst being relatively benign on IEC-18 cells.LC and Lactobacillus and Bifidobacterium spp. are able to change the expression of genes involved in cell death, apoptosis, metastasis, and cell proliferation by several studies [Lactobacillus casei ATCC 393 induced apoptosis of colon carcinoma cells by up regulating the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) protein which, in turn, decreased tumor incidence in mice [L. rhamnosus GG on normal IEC-18 cell line [Several other studies have also confirmed the apoptotic effects of different strains of probiotics in cancer cells and animal models , 30, 31. studies . Tiptiri in mice . In addiell line .L. reuteri, were more effective than trastuzumab (15% primary apoptosis) in terms of apoptosis induction on LS174T cells and all, except BC, had generally less apoptotic activity than cetuximab.Moreover, BC had a notable result among other bacterial groups with similar effect as cetuximab (25.02% primary apoptosis) but had a significantly higher influence than trastuzumab on apoptosis induction on LS174T cells after 120h incubation (P = 0.001). In addition, all of the bacterial groups, except Since cetuximab and trastuzumab bind to the outer membrane section of EGFR and HER-2 proteins, it is believed that the signaling responses triggered by activation of these two receptors result in apoptosis of cancer cells –35.It might be that the signaling pathways triggered by cetuximab- EGFR binding and BC- cell attachment are equally strong in apoptosis induction. The reason of higher apoptotic effect of bacterial groups compared with trastuzumab could be that they affect multiple signaling pathways concomitantly (in addition to HER-2 and EGFR pathways) for apoptosis induction which make the total triggered signal stronger than trastuzumab. In addition, bacteria as living organisms have the ability of quorum sensing their environment, which probably help them synergistically activate stronger apoptotic signals in the cell. It may prove that a combination of Bifidobacterial strains have a stronger quorum sensing, compared to other tested bacteria, in response to attachment to cancer cells and hence, the higher apoptotic activity of BC compared to other bacterial groups.The low expression of EGFR and HER-2 receptors in normal cells compared to LS174T cells , 36  but also on the characteristics of the bacterial treatments since, for instance, BC had the highest apoptosis rate in LS174T cells compared to other bacteria, but the lowest rate in IEC-18 cells. One of these bacterial characteristics could be surface proteins and the attaching power to different receptors which are different among bacteria.In this study, BC had significant EGFR-down regulating effects among LS174T cells, both at mRNA and protein levels, whilst having the least interruption on IEC-18 cells compared to other treatments (in terms of down regulating this onco-marker). These properties make BC a very efficient anti-EGFR treatment which significantly reduces EGFR in cancer cells whilst having no significant impact (regarding EGFR levels) on normal cells. LC, on the other hand, had no significant effect on LS174T in terms of reducing the EGFR expression (neither on the mRNA nor the protein levels), but significantly decreased the gene expression in normal cells during 120h incubation time; therefore, LC seems not efficient in this regard.The rationale for all this should again come from the fact that the plethora of surface proteins differ among bacteria/ and strains of bacteria which makes their attaching power to variant cell types different.EGFR levels. One result to be noted is that all the bacterial groups had significantly less interruptions in the EGFR expression among normal IEC-18 cells, compared to cetuximab and trastuzumab. Since Lactobacilli and Bifidobacteria are the commensal flora of the gut in many living organisms, it seems logical that they have less interruptions in normal epithelial cells compared to the drugs.There were no preferences between the three bacterial treatments in decreasing https://www.kegg.jp). If we assume that the signals triggered by the heterodimers finally regulate the expression of both members of the heterodimer compartment, then the bacteria might have a higher chance of down regulating HER-2 (compared to EGFR/ErbB-1) in the cell. BC was the strongest bacterial treatment in down regulating HER-2 in LS174T cells (the same strength as trastuzumab and only second to cetuximab) but did not significantly change this tumor marker’s expression among IEC-18 cells (in contrast to the drugs). LC had a good effect on cancer cells in terms of down regulating HER-2, but it significantly increased the expression of HER-2 among IEC-18 cells (as did the drugs). Therefore, LC might again have anti-HER2 properties of questionable value. The reason why HER-2 expression among IEC-18 cells has unexpectedly increased by the treatment groups in this study remains unclear.Compared to EGFR, HER-2 (mRNA and protein levels) was much more efficiently inhibited by the bacterial groups in LS174T cells. Several receptors and factors on the cell might be affected by these bacteria that all end up influencing the expression of HER-2 in the cell. Among the ErbB family receptors, HER-2 (ErbB-2) has a higher capacity to form heterodimers with other family members  . In the meanwhile, BC did not have a notable effect on PTGS -2 expression among normal IEC-18 cells (significantly less interruptive than the drugs). For all these reasons, BC was again the most effective treatment, compared to other bacterial groups, in decreasing COX-2 in cancer cells while not having a great impact on this gene among normal cells.The bacterial groups had a higher effect on down regulation of COX-2 (mRNA and protein levels) compared to EGFR and, to a lesser extent, to HER-2. BC, with 20 folds decrease in PTGS-2 down regulation among LS174T cells, compared to cetuximab and trastuzumab. This might be an expected observation since the PTGS-2 down regulating effects of some probiotic strains have already been reported by several studies [PTGS-2 gene and COX-2 protein. Besides, there are studies which have reported an association between the expression of COX-2 with HER-2 [PTGS-2 gene in LS174T and also in IEC-18 cells. This might suggest an indirect influence of cetuximab and trastuzumab on COX-2 expression and might affect the signaling pathways in which COX-2 is involved.Another interesting remark in this study was that all the bacterial groups, except LC, were stronger in  studies , 17, whith HER-2  and EGFRth HER-2 , as wellth HER-2 . Cetuximin vivo experiments in this study. BC significantly ameliorated the DAI, almost completely restored the colon length, inhibited the increase in tumor incidence and prevented the progress of tumors to higher stages and grades.The beneficial effects of BC on CRC- prevention was proved by Bifidobacterium lactis synbiotically combined with resistant starch significantly prevented CRC among rat-AOM model as well as facilitating apoptotic deletion of carcinogen-damaged cells in rat colon [Bifidobacterium longum combined with lactulose significantly suppressed AOM-induces colonic crypt foci which are preneoplastic markers [Studies by Le Leu et al. showed that the probiotic at colon , 7. In a markers .All these results confirm the preventive role of BC in CRC occurrence among CRC animal models.EGFR, HER-2 and PTGS-2 (COX-2) onco-markers and significantly ameliorate disease activity index, restore colon length, inhibit the increase in tumor incidence and prevent the progress of tumors to higher stages and grades.Overall, considering the effects on both the cancer and normal cell lines, Bifidobacteria cocktail is the most efficient treatment, compared to other bacterial combinations used in this study. This potential probiotic has considerable “protective” anti-cancer properties comparable to the already in use drugs cetuximab and trastuzumab and is able to concomitantly down regulate In general, this potential probiotic could be considered as a suitable nutritional supplement to be used along with the drugs cetuximab and trastuzumab to treat and prevent CRC.Since the results of this study is probably strain- and cell type- specific, it is recommended that more variants of Bifidobacterial strains and cell types be investigated to obtain a more comprehensive conclusion on the anti-CRC mechanisms of action of this bacterium.S1 FigEffects of the bacterial groups on LS 174T cancer cells (A); cetuximab and trustuzumab drugs on LS 174T cancer cells (B); bacterial groups on IEC-18 primary cells (C); cetuximab and trustuzumab drugs on IEC-18 primary cells. Results were expressed as mean; error bars (SD); n = 3. Statistical analysis was performed using one-way ANOVA test. * indicates P-values less than 0.05, ** indicates P-values less than 0.01, and *** indicates P-values less than 0.001. Untreated cells were used as controls.(TIF)Click here for additional data file.S2 FigCells considered as viable were Annexin V and PI negative; cells in early apoptosis stage were Annexin V positive and PI negative; and cells in late apoptosis/ necrosis stage were both Annexin V and PI positive. Untreated cells were used as negative controls and cetuximab and trastuzumab were used as positive controls.(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S1 Raw images(TIF)Click here for additional data file.


To evaluate the value of the 3-h post-ERCP serum amylase level for early prediction of post-ERCP pancreatitis (PEP).A study of 206 patients performed ERCP (Encoscopic Retrograde Cholangio-Pancreatography) at a single centre was done from Jan. 2011 to Nov. 2016. The serum amylase or lipase level was measured at 3 h after ERCP. The patients with PEP were recorded. ROC curves were used to statistically analyze the data: The enrolled patients were divided into two groups according to gender, then we analyzed the data respectively. We comprehensively evaluated the predictive value of PEP by 3-h post-ERCP serum amylase level based on the results above.P < 0.001). The standard error (SE) was 0.0507, the 95% confidence interval (CI) was 0.756–0.866, and the optimal cut-off value was 351 U/L . Of the 206 patients, there were 83 patients with both 3-h post-ERCP amylase level and lipase level detected, and the ROC curves were plotted for both serum amylase and lipase respectively. The ROC curve matched-pair testing was carried out: The areas under the ROC curves were statistically significant. (P < 0.001) The area under the ROC curve for the 3-h post-ERCP lipase was 0.778, the 95% confidence interval was 0.673–0.862, and optimal cut-off value was 1834 U/L. The area under the ROC curve for the 3-h post-ERCP serum amylase was 0.780, and the 95% confidence interval was 0.676–0.864. The optimal cut-off is 380 U/L, and there was no statistically significant difference between the two for diagnostic accuracy. According to gender, 206 patients were divided into 2 groups, and the ROC curves were drawn respectively. Based on statistical analysis, there was no statistically significant difference in the diagnostic accuracy of the two groups. In the male group, 436 U/L serum amylase provided the greatest diagnostic accuracy with sensitivity (SE) of 70.5%, specificity (SP) of 89.2%, positive predictive value (PPV) 87.5%, and negative predictive value (NPV) 78.1%. Whereas, in the female group, 357 U/L serum amylase provided the greatest diagnostic accuracy with sensitivity of 76.9%, specificity of 81.2%, positive predictive value of 80.4%, negative predictive value of 77.9%.Two hundred six patients  were enrolled. PEP occurred in 21 patients (10.19%) among them. The median time to discharge was 7 days  after the procedure. In the 206 patients, the 3-h post-ERCP pancreatic amylase level was used as the test variable, and the PEP occurrence as the state variable to plot the ROC curve. The area under the curve (AUC) was 0.816, and was statistically significant (1. The 3-h post-ERCP serum amylase level is a useful measurement for predicting post-ERCP pancreatitis. 2. There was no significant difference between serum amylase and lipase 3-h post-ERCP for predicting PEP. 3. There was no statistically significant difference between male and female using the 3-h post-ERCP serum amylase level to predict PEP. For female, the optimal cut-off value was 357 U/L, whereas male 436 U/L . Since first reported by McCunne in 1968 . HoweverFrom January 2011 to November 2016, a total of 546 patients were diagnosed or treated with ERCP at the First Affiliated Hospital of Dalian Medical University. The study was conducted in compliance with the Helsinki Declaration and in accordance with local legislation, was approved by the Ethics Committee of First Affiliated Hospital, Dalian Medical University. (Ethics References No: YJ-KY-FB-2019-01). Written informed consent was obtained from all of the patients or their relatives before the procedure.The inclusion and exclusion criteria were as follows:Inclusion criteria: (1) preoperative serum amylase and lipase levels were normal; (2) age ≥ 18 years; (3) serum amylase level was measured 3 h after ERCP; (4) patients have not used trypsin inhibitor before the diagnosis of PEP.Exclusion criteria: (1) preoperative diagnosis of acute and chronic pancreatitis (2) abnormal renal function (serum creatinine >92umol / L) (3) pregnant womenAccording to the inclusion and exclusion criteria above, a total of 206 patients were enrolled, including 92 males and 114 females, among them, 84 cases were simultaneously detected for serum lipase.Acute pancreatic abdominal pain within 24 h after ERCP;Serum amylase more than 3 times the upper limit of normal within 72 h after ERCP  or lipase greater than 3 times the upper limit of normal within 96 h after ERCP ;Contrast-enhanced CT, MRI, abdominal ultrasound showing acute pancreatitis changes ; having two of the three criteria will lead to a diagnosis of PEP.The necessity for new or continued hospitalization for at least 2 nights.The diagnosis of pancreatitis after ERCP is based on the consensus reached by Cotton et al. , 13, andDuodenoscope , guide wire , triple lumen sphincterotome , CRE balloon catheter , retrieval balloon catheter , Dormia basket , Percuflex Biliary Stent , nasal biliary drainage tube , WallFlex Biliary RX Fully Covered Stent System ,Mechanical lithotripsy .Some ERCP procedures were accomplished under ECG monitoring. Tetracaine was given for local anesthesia of the pharynx. The patients were sedated and relieved pain by intramuscular administration of diazepam and meperidine. Twenty mg of butyl scopolamine bromide was injected intramuscularly prior to the procedure to inhibit duodenal peristalsis.A sphincterotome with a guide wire was used for selective cannulation. Difficulties in selective cannulation including stenosis and sclerosis of the papillary, incarceration of common bile duct (CBD) stone at papilla, and periampullary diverticulum, especially the papilla is located at bottom of the diverticulum.For some cases difficulty in bile cannulation, precut through pancreatic duct was applicated. For cases with incarceration of CBD stone at papilla, needle knife was used for precut sphincterotomy. For papilla located at the bottom of diverticulum, it was exposed by eversion diverticulum through biopsy forceps, or fixation by metal clip, or submucosal injection of saline.For patients with CBD stones, small endoscopic sphinecterotomy (EST) alone or EST combined with endoscopic papillary balloon dilation (EPBD) was performed. For biliary tract benign stricture or biliary fistula, ERBD was given. For biliary or pancreatic malignant tumor, a self-expanding metal stent was placed.Mechanical lithotripsy was used for large common bile duct stones. However, if patient was in poor condition and cannot tolerate long procedure time, just ERBD was placed for drainage and to relieve symptoms. And a next ERCP was performed for removal of cholelithiasis 3 months later.Serum amylase, lipase, biochemical liver function and renal function were detected by Johnson & Johnson’s VITROS FS5.1 automatic biochemical analyzer.According to the inclusion and exclusion criteria above, a total of 206 patients were enrolled, and each patient’s progress note was reviewed by the medical record system. Sudden pancreatitis-like mid-abdominal pain within 24 h after ERCP was marked positive, otherwise negative; combined with CT, serum amylase, lipase levels, comprehensive estimation was made according to the PEP diagnostic criteria .1. Descriptive analysis of the enrolled patients, a preliminary understanding of the basic situations of the study objects. 2. The ROC curve was plotted according to the 3-h post-ERCP serum amylase level of the enrolled patients and PEP diagnosis. According to the area under the ROC curve and the 95% confidence interval of the area, the accuracy of the tests was evaluate based on statistical tests. The Youden index was calculated according to the sensitivity and specificity of each level and the optimal cut-off value was obtained. The sensitivity, specificity, positive and negative predictive value, positive and negative likelihood ratio, Youden index of different cut-off sites were calculated, comprehensively evaluated the accuracy and predictive value of the predictive diagnostic method. 3. According to gender, they were divided into two groups, and the ROC curves were drawn and analyzed separately aimed to find best cut-off in different gender. As a same lab test whose diagnosis accuracy (be showed as AUC in ROC) should have no difference between genders or other classifications of subjects. So we use Z test to test the reliability of data, then obtain the optimal diagnostic cut-off value according to these two curves. 4. Matched-pair analysis of ROC curves in patients who were simultaneously tested for both serum amylase levels and lipase levels 3 h after ERCP. According to the two test Methods, the ROC curves were drawn and statistically tested, and the difference test (Z test) was performed to compare and analyze the diagnostic accuracy and diagnostic value in the two groups.P value was looked up in the table .Medcalc was applied to perform matched-pair analysis. The ROC curve of serum amylase of different gender patients were plotted using SPSS software, and then the difference test was performed according to the standard error and the area under the curve. The Z value was manually calculated, and the Related formula:Sensitivity: Se; Specificity: Sp.A total of 562 patients who underwent diagnostic or therapeutic ERCP in our hospital between January 2011 to November 2016. The total number of patients who met the criteria for enrollment was 206, 92 males and 114 females. Among the 206 patients, endoscopic sphinecterotomy  was performed in 136 patients; endoscopic nasobiliary drainage (ENBD) was performed in 140 patients; endoscopic retrograde biliary drainage (ERBD) was performed in 36 patients .P < 0.0001. The area under the ROC curve was statistically significant, and the optimal cut-off value was 351 U/L. The sensitivity, specificity, positive and negative likelihood ratio, positive and negative predictive value, and Youden index of different cut-off values are shown in Table The non-parametric method was used in this paper, the serum amylase level 3-h post-ERCP as the test variable, the PEP occurrence as the state variable and the ROC curve was drawn. The result is shown in Fig. As seen from the Table In order to investigate whether different genders groups need different diagnostic criteria, ROC curve was applied to evaluate this issue in our study. Two hundred six patients were divided into two groups according to gender, and the ROC curves were plotted respectivelyFig. , female.P = 0.001 for both groups), the area under the curve for the male group was 0.859, the standard error was 0.065, and the area under the curve for the female group was 0.794, and the standard error was 0.068 . The area under the ROC curve of lipase levels 3-h post-ERCP was 0.778, with a 95% confidence interval of 0.673–0.862; The area under the ROC curve of amylase 3-h post-ERCP was 0.780, and the 95% confidence interval was 0.676–0.864, indicating that the accuracy of predicting PEP was high   Table .Fig. 4RERCP has become an important method in clinical diagnosis and treatment for biliary and pancreatic diseases. However, its complications should not be ignored, especially post-ERCP pancreatitis (PEP). As the most common post-ERCP complication, PEP has restricted the development of ERCP. The average incidence of PEP is reported to be 3.5% in the literatures, but in high-risk groups , the incidence can be as high as 30% . Since ip = 0.007) through a retrospective study of 1631 patients. Kapetanos et al. [The role of serum amylase levels in predicting the occurrence of PEP after ERCP has been recognized . Takayoss et al.  also havs et al. . The stus et al.  suggest Our study showed that the serum amylase level 3-h after ERCP has a good accuracy in the prediction of PEP. The cut-off at 351 U/L provides the optimal diagnostic accuracy, when setting the cut-off at 105 U / L , the negative likelihood ratio can reach 0.21, indicating that this is a valuable predictive value for eliminating PEP.Studies have confirmed that female gender is one of the important risk factors for pancreatitis after ERCP , so whetSerum lipase is a specific diagnostic indicator of pancreatitis. Its activity is maintained longer than amylase, and there is no other source in the blood , so it iHowever, this was a retrospective study in a single center, and small sample was another limitation for the study. More cases are required to confirm the value of the 3-h post-ERCP serum amylase level for early prediction of post-ERCP pancreatitis in the future.In summary, we have come to the following conclusions:The 3-h post-ERCP serum amylase level is a useful measurement for early predicting post-ERCP pancreatitis. With the 3-h post-ERCP serum amylase level 1–1.5 times upper limit of normal, the negative predictive value is high.There was no significant difference between serum amylase and lipase 3-h post-ERCP for predicting PEP.There was no statistical difference between male and female using the 3-h post-ERCP serum amylase level for prediction PEP. For female, the optimal cut-off value was 357 U/L, whereas male 436 U/L.


The extracellular matrix (ECM) composition greatly influences cancer progression, leading to differential invasion, migration, and metastatic potential. In breast cancer, ECM components, such as fibroblasts and ECM proteins, have the potential to alter cancer cell migration. However, the lack of in vitro migration models that can vary ECM composition limits our knowledge of how specific ECM components contribute to cancer progression. Here, a microfluidic model was used to study the effect of 3D heterogeneous ECMs  on the migration distance of a highly invasive human breast cancer cell line, MDA-MB-231. Specifically, we show that in the presence of normal breast fibroblasts, a fibronectin-rich matrix induces more cancer cell migration. Analysis of the ECM revealed the presence of ECM tunnels. Likewise, cancer-stromal crosstalk induced an increase in the secretion of metalloproteinases (MMPs) in co-cultures. When MMPs were inhibited, migration distance decreased in all conditions except for the fibronectin-rich matrix in the co-culture with human mammary fibroblasts (HMFs). This model mimics the in vivo invasion microenvironment, allowing the examination of cancer cell migration in a relevant context. In general, this data demonstrates the capability of the model to pinpoint the contribution of different components of the tumor microenvironment (TME). Breast cancer is among the three most common cancers worldwide and is the most common malignancy in women ,2. CurreThe tumor microenvironment (TME) has emerged as a critical player in cancer progression and metastasis ,6,7. TheTraditional in vitro models for studying cell migration include wound healing assays and transwell assays, and present several limitations ,23. For In this work, we incorporated specific TME components  in a relevant 3D platform to build the complexity of the TME. Specifically, we mimicked an invasive stage of breast cancer surrounded by a complex and relevant TME using a microfluidic platform previously developed in our laboratory. This platform enables the creation of a lumen structure within a 3D collagen matrix, which is easily modified to include different fibroblasts and ECM compositions. Based on in vivo proteomics literature ,43,44, wUsing a previously described microfluidic called LumeNEXT, we sought to mimic an invasive stage of breast cancer and investigate the impact of ECM and stromal cell interactions on cancer cell invasion a. Using We also performed a characterization of the cells used for this model. First, we confirmed the expression of GFP in 97% ± 2.3% of the MDA-MB-231s . In addiTo characterize the interaction between the fibroblasts and cancer cells, we used a custom co-culture 96-well plate . The use of this plate allows the formation of a liquid bridge between adjacent wells, connecting the two cells populations cultured in each well via soluble factors . Brieflyp = 0.0019) (p = 0.0063) (p = 0.0015) (p = 0.0365), compared to HMF. To determine whether CAFs secrete more fibronectin than HMFs, the expression of fibronectin in CAFs and HMFs cultured in 3D collagen matrices was assessed via Western blot. As anticipated, fibronectin expression was significantly increased in CAFs as compared to HMFs  with embedded fibroblasts  for 48h a–c. Canc 0.0019) d. In the 0.0063) d. When c 0.0063) b,c. In t 0.0015) e. Howeve 0.0015) e. When c to HMFs f,g. Wholp = 0.0351, p = 0.0101 and p = 0.0121, respectively). On the other hand, in co-culture with CAFs, a significant increase in MMP-3 was found for the collagen matrix (12-fold) and the fibronectin-rich matrix (14-fold)  and, a significant increase in MMP-9 (3-fold) within a fibronectin-rich matrix (** p = 0.0084)  b–d.In order to determine if matrix composition had an impact on cell MMP secretions, we calculated the fold change of the fibronectin-rich matrix over the collagen matrix for the different MMPs e–g. In t2 for the collagen matrix and 416.0 ± 40.25 μm2 for the fibronectin-rich matrix, revealing a significant increase in the gap area in the fibronectin-rich matrix (* p = 0.05)  imaging was performed after 48 h of culture a. Eviden = 0.05) f. In thematrices f.The gaps observed in the matrix were indicative of matrix degradation, and only minimal gaps were observed in the MDA-MB-231 monocultures . In the p = 0.0069) (p < 0.0001) (p < 0.0001)  was used. All cultured conditions were treated with 30 µM marimastat or vehicle control (dimethyl sulfoxide—DMSO) for 48 h and then were imaged a. Then,  0.0069) b. In con 0.0069) b. In co- 0.0069) c, reveal 0.0001) d. Simila 0.0001) d.p < 0.0001), 357.9 ± 108.9 µm for the vehicle treatment compared to 201.6 ± 67.5 µm for the marimastat treatment (p < 0.0001)  . The dec 0.0001) . Interes 0.0001) . However 0.0001) .In recent years, the TME has emerged as a highly regulative factor of cancer progression . It is wTo better understand cancer cell migration within different tumor-like microenvironments, we used a 3D microfluidic model that recapitulates different microenvironments. Using this model, we sought to investigate the effect of fibronectin-rich matrices and fibroblasts on MDA-MB-231 cancer cell migration. To generate the different stromal components of the model, we used normal (HMFs) and cancer-associated fibroblasts (CAFs). The identification of the cancer-associated phenotype was corroborated via podoplanin staining . The staIn addition, we sought to characterize the interactions among the cancer cells and fibroblasts via soluble factor crosstalk. This characterization revealed that cancer cells crosstalk with HMFs resulted in an increase in MDA-MB-231s growth rate. Interestingly, these results are in agreement with recent literature demonstrating that normal breast fibroblasts that secrete high amounts of CXCL12, which is characteristic of our HMFs, induce MDA-MB-231 proliferation via the CXCR4 receptor . On the After the characterization, cancer cells were seeded in the different matrix compositions  with embedded fibroblasts  for 48 h. We observed that the number of MDA-MB-231 cells migrated into the matrix was significantly higher for the fibronectin-rich matrix, both with CAF and HMF, as compared to the collagen-only control. When we evaluated the average migration distance for the collagen-only matrices, cancer cell migration increased in the cocultures with CAFs, as compared to the co-cultures with HMFs. Interestingly, in the presence of HMFs, a normal component of the TME, fibronectin induces changes in the fibroblasts that, in turn, enhance the migration of the cancer cells. These results suggest that within a normal microenvironment (HMFs), a fibronectin-rich matrix is acting as a tumor-promoting factor, “educating” the normal environment to support cancer progression. On the other hand, we did not observe this effect in the presence of CAFs. However, it is known that CAFs secrete more fibronectin than HMFs, as demonstrated in In breast cancer, tumor progression and metastasis have been found to be driven by matrix metalloproteinases (MMPs) shaping of the TME ,59. In tIt is well recognized that fibroblasts can interact and communicate with the surrounding ECM, resulting in ECM structure remodeling. The remodeling of the ECM is regulated, in part, through the secretion of matrix-degrading enzymes , which are known to facilitate cancer invasion through degradation of the ECM. Therefore, we next studied the presence of collagen disruptions in the collagen using second harmonic generation imaging. This study revealed an increase in the distribution of matrix gap areas in fibronectin-rich matrices, where HMFs were co-cultured with cancer cells. This effect is consistent with the increase in MMP-2, -3, and -9 secretion in previous figures. However, the gap area in the co-culture with CAFs did not change in the fibronectin-rich matrix as compared with the collagen matrix. Interestingly, these MMPs can degrade fibronectin and not collagen I, suggesting that the fibronectin matrix is signaling the HMFs to produce MMPs that can degrade the surrounding matrix. As for the degradation of collagen I, these MMPs have the potential to activate other MMPs to degrade collagen I. To investigate if the degradation of the matrix was responsible for changes in MDA-MB-231 migration, we used a broad-spectrum MMP inhibitor: Marimastat. Marimastat inhibits MMP-2, MMP-9, MMP-1, and other MMPs and, it has been reported to inhibit cancer cell migration 3D in vitro at a 30 µM concentration  and to iInterestingly, MDA-MB-231 monoculture and in co-culture with CAFs showed the largest migration distance. Therefore, we hypothesized that migration must occur in a mechanism independent of MMP-2, -3, and -9 in these conditions. These results do not match the previously described results, which demonstrated the efficacy of the inhibitor in vitro ,63. HoweAltogether, our microfluidic 3D in vitro model allowed us to mimic important features of the breast tumor microenvironment by including only a few key components of the tumor microenvironment. Using this model, our findings demonstrate the utility of our model to study the contribution of different microenvironment components  on breast cancer cell migration. The usefulness of this model could be further improved by incorporating different cell types representing distinctive types of cancers, therefore providing researchers with a powerful tool to advance cancer research. As an example, triple-negative breast cancer (TNBC), a highly aggressive and migratory cancer type was represented in this study by using the cell line MDA-MB-231. Therefore, the incorporation of cell lines that represent the different molecular subtypes of breast cancer using this model could provide further insights into cancer progression. Further, we could test the relevance of this model by including other cancer types in the model and by using patient-derived cancer cells. In the future, we envision the current platform as a screening tool to test the influence of other matrix proteins that are found in vivo . Overall2. All cells were cultured to 90–95% confluency for all experiments, and the media was changed every 2–3 days.Human mammary adenocarcinoma cells MDA-MB-231 were selected for high invasiveness and their ability to metastasize in vivo . For allLumeNEXT fabrication was performed as previously described . BrieflyTo achieve maximum hydrogel adhesion to the device chamber, a 2-step coating of 2% poly(ethyleneimine)  diluted in deionized DI water for 10 min was loaded into the side ports. The PEI solution was aspirated, and 0.4% glutaraldehyde  diluted in deionized (DI) water was loaded into the side ports and incubated at room temperature for 30 min. During the GA incubation, the collagen solution was prepared on ice refer to . After tHigh-density rat-tail collagen type 1  was diluted with 5X PBS (diluted with DI water from the 10× stock described before) and neutralized with 0.5 M NaOH  achieving a final concentration of PBS 1×, and a pH of 7.4. This mixture was diluted with fibrinogen  and media to achieve a final concentration of 2.25 mg/mL. For the collagen solution containing fibronectin, fibronectin solution  was added to a final concentration of 100 μg/μL in the collagen. For experiments with stromal cells in the matrix, HMFs or CAFs, a final concentration of 500 cells/μL was added to their respective collagen solution. Immediately after washing, 8 µL of collagen solution was loaded through the side ports and polymerized at room temperature for 10 min. Finally, a small droplet of media (5 μL) was placed on top of the side ports to prevent evaporation, and devices were transferred to 37 °C for 1 h to allow collagen to polymerize fully.After incubation, a droplet of media (5 μL) was added to the input port under sterile conditions. To remove the PDMS rod, the rod was pulled through the output port using isopropanol-sterilized tweezers. This procedure yielded a hollow lumen structure within the collagen matrix filled up with media and ready for cell loading. All fluid handling procedures were conducted with standard pipettes, uniquely enabled by passive pumping . A 1.5mg® Eclipse inverted microscope  and processed using the National Institutes of Health ImageJ software. To analyze the migration distance, each Z-stack was Z-projected, and the MDA-MB-231 distance from the lumen was quantified using Image J [The GFP signal from the MDA-MB-231 was used to track cancer cell migration out of the lumen. For each device, Z-stack imaging was performed after 48 h of culture. Bright-field images were acquired at 4X using a Nikon TI Image J . Multiplexed protein secretion analysis was performed on the cancer-fibroblasts co-cultures, cancer monocultures, and fibroblast monocultures in both types of matrix composition. The analysis was performed using the Magnetic Bead-Based Multiplex ELISA system MAGPIX  using a Human MMP Magnetic Panel (3-Plex)  as described elsewhere . CollectCollagen hydrogel structure was visualized by second harmonic (SHG) using a custom-built inverted multiphoton microscope , as described previously . The sysSHG images from the different collagen matrices were analyzed using the Diameter J plugin for ImageJ. Briefly, image segmentation was performed for each image following binarization. All gap areas in the image were automatically measured with this plugin.For experiments in which cells were fluorescently labeled with red or blue cell tracker, stock solutions of cell tracker red CMTPX  and blue  were prepared following supplier instructions. Then, the stock solution was diluted 1:1000 in the growth medium. Cells were trypsinized and incubated in the cell-tracker diluted medium for 30 min. Finally, cells were washed twice with 1X PBS to remove the excess of the cell tracker.For immunofluorescence staining, cells were washed with PBS for 30 min between each step. Unless specified otherwise, steps took place at room temperature. Washing buffer  and blocking buffer  in 0.1% PBS-Tween 80) were made in advance and stored at 4 °C until use. Cells were fixed with 4% paraformaldehyde (PFA)  for 15 min, then incubated with 0.2% Triton® X-100  for 30 min for permeabilization. Finally, devices were incubated with 10 µL of blocking buffer at 4oC overnight. Texas Red-X Phalloidin  and DAPI  were used to stain actin cytoskeleton and nuclei, respectively. An anti-fibronectin antibody was used to stain the presence of fibronectin in the collagen gel . For activated fibroblasts characterization, an anti-fibroblast activation protein antibody  and an anti-podoplanin antibody . Fluorescent images were acquired at 10× using a Nikon TI Eclipse inverted microscope (Melville) and processed using the National Institutes of Health ImageJ software.5 cells were embedded in a final 1 mL volume of 3 mg/mL type-I collagen hydrogel. Cells were cultured for 72 h, gels were washed 3× with PBS prior to lysis in 2× RIPA [Cell lysates from HMFs or CAFs that were cultured in a 3D collagen matrix were analyzed for the expression of fibronectin. To ensure adequate protein concentration for western blot analysis, 2 × 10 2× RIPA  and 5× LMarimastat  was used to inhibit the activity of different MMPs, such as . A stock solution was prepared at 10 mM concentration following supplier instructions. For the treatment with marimastat in the different microenvironments, the stock solution was diluted to a final concentration of 30 µM marimastat. Treatment with 30 µM Marimastat or vehicle control (DMSO) was performed for 48 h, following imaging acquisition. Image analysis was performed to determine changes in migration distance, as described in previous sections.p < 0.05. One-to-one comparisons were performed with an unpaired Student t-test with Welch’s correction (if SDs were inhomogeneous) after the normal distribution was proved via the Shapiro-Wilk test. If the normality test was not passed, a non-parametric test was performed (Mann-Whitney test).All the experiments were repeated at least 3 times as independent biological replicates. All results were presented as the mean ± standard deviation of the mean. Data were analyzed using GraphPad Prism 8 , and statistical significance was set at The microfluidic model presented in this paper mimics the in vivo invasion of cancer cells within a 3D microenvironment that includes relevant fibroblasts and extracellular matrix composition. We found that a tumor-like matrix, such a fibronectin-rich matrix altered the normal mammary fibroblasts over the cancer-associated fibroblasts. Some of the changes observed were an increase in the number of migrating cells, longer migration distance, higher secretion of MMPs, as well as a resulting increase in matrix remodeling. In co-culture with HMFs, MMP inhibition was effective in inhibiting cancer cell migration in the collagen matrix and not in the fibronectin-rich matrix. In addition, MMP inhibition was performed in cancer cells monocultures and was found to be effective regardless of the matrix, which is consistent with the literature. In conclusion, without incorporating fibroblasts, a highly MMP-secreting cell type, MMPs inhibitors are highly effective. Therefore, the lack of incorporation of this component in in vitro studies could explain the limited successes of MMP inhibitors in clinical trials, which further suggests the need to develop alternate methods to specifically target fibroblast induced matrix remodeling. Examples of ongoing CAF anti-cancer therapy include CAF specific depletion with anti-FAP antibodies , maintai


Although acute gastroenteritis (AGE) has been reported as a common infectious disease in children, there is scarce information about enterovirus (EV) circulating associated with AGE cases in Brazil. The purpose of the present study was to identify and characterize the enteroviruses associated with AGE in children in Belém, Brazil. A total of 175 stool samples were obtained from children hospitalized revealing the presence of EV in 26.3% (46/175) of infections. EV type was identified in 78.3% (36/46) and EV-B species  was the most prevalent EV-detected followed by EV-C  and EV-A . This study has provided important information about the enterovirus circulation in Pará state, Northern Brazil. Picornaviridae family, such as Parechovirus, Cosavirus, Salivirus and Aichivirus have been identified as agents associated with diarrhea in humans [Picornaviridae family also has the Enterovirus genus, whose association with AGE has been recognized and reported in many studies [Acute gastroenteritis (AGE) is one of the most common diseases in humans, mainly in children and remains as important cause of morbidity and mortality among infants around the world . Children humans , 9. The  studies , 7, 10. In Brazil, a limited number of studies on EV that are associated with AGE have been reported , 12. TheFrom May 2010 through April 2011, 175 stool specimens were collected from children (< 5 years) who had been suffering from AGE and attended to the Pediatric Clinic of Pará. Viral RNA was extracted  directly from the clinical specimens and initially subjected to a broad-reactive real time RT-PCR (rRT-PCR) for human enteroviruses as previously described , 14. EV-After inoculation, the specimens that did not have effect in the production of CPE  were submitted to a semi-nested PCR (RT-snPCR) amplification of partial VP1 gene according to previously described , 16. EV-CVB3, E7 and E9 were the most frequently detected type from the EV-positive specimens Table . FurtherIn this study, 26.3% (46/175) of specimens analyzed were EV-positive. Similar results were obtained from studies performed in India, which reported an EV detection rate of 33–40% . SurprisTo further analyze EV types identified in AGE patients, we carried out a phylogenetic analysis based on partial VP1 sequences  in areas of low vaccine coverage and deficient acute flaccid paralysis surveillance. Additionally, in the context of global eradication of polioviruses, information on non-polio enteroviruses circulation is key to understand their role in AGE context and other enteroviruses infections-associated. The difficult in the access the patient records and to survey other causative agents involved in AGE represented a study limitation.